SWISLOCKI AND TIERNEY
Solubilization, Stabilization, and Partial Purification of Brain Adenylate Cyclase from Rat+ Norbert I. Swislocki* and Joan Tierney
ABSTRACT:Membrane-bound adenylate cyclase from rat brain has been solubilized with Lubrol-PX. The soluble enzyme is nonsedimentable at 165,OOOg for 2 hr, is filterable through 0.22 p Millipore filters and is responsive to F-. The solubilized adenylate cyclase has been stabilized for at least 7 days by the addition of 5 mhi N a F and 5 mM MgS04. The enzyme has an estimated molecular weight of 800,000 which was obtained on the basis of a calibrated Bio-Gel A-15 m (agarose) column. The enzyme has been purified 18-fold representing the removal of over 99 of the starting protein. Addition of fractions removed during purification did not increase enzyme activity. The partially purified adenylate cyclase has ATPase and adenosine 3 'J '-cyclic monophosphate phospho-
A
denylate cyclase catalyzes the conversion of adenosine triphosphate to cAMP.1 and plays a pivotal role in mediating the effects of a wide variety of hormones (Sutherland et a/.. 1965). The physiological importance of adenylate cyclase has long been recognized and its properties as a particulate, hormone-responsive membrane-associated enzyme have been extensively described and are the subject of investigation (Bitensky et a/.. 1968; Chase and Aurbach, 1968; Pohl et a/., 1971). Extensive purification of adenylate cyclase has been hampered by its resistance to solubilization and its lability during storage and processing. Levey's (1970) success in solubilizing myocardial adenylate cyclase with Lubrol-PX, a nonionic detergent, stimulated our efforts to solubilize, stabilize, and purify adenylate cyclase from the brain, a tissue rich in adenylate cyclase activity. A portion of these studies has been presented in abstract form (Swislocki, 1972). Methods Adenylate cyclase activity was measured by the method of Krishna et a/. (1968). Incubations were carried out at 30" for 15 min in 0.04 M Tris-HC1 (pH 7.4), containing 1 mhi ATP, 1 mhi MgCI?, and 10 mM theophylline. Each reaction contained 2-3 pCi of [a3*P]ATPobtained from International Chemical and Nuclear Corp. After incubation 0.5 mg of cAMP was added to each tube and reactions were terminated by boiling for 3 min. [cY-~~PIcAMP was purified by Dowex 50 (H+)chromatography and ZnS04-Ba(OH)? precipitation (Krishna et a / . , 1968). Recovery of labeled cAMP was followed spectrophotometrically at 260 nm of added CAMP.
t From the Sloan-Kettering Institute for Cancer Research, New York, New York 10021. Receiued December 7, 1972. This investigation w a s supported i n part by Research Grant CA-08748 of the National Institutes of Health and Grant GB-19797 of the National Science Foundation. Abbreviation used i s : CAMP,adenosine 3 ',5'-cyclic monophosphate. Lubrol-PX is a n ethylene oxide condensate of dodecanol.
1862
BIOCHEMISTRY VO , L . 12,
NO.
10, 1 9 7 3
diesterase activities associated with it. The material obtained after ion exchange chromatography and gel filtration has low mobility on electrophoresis in acrylamide gels. Electrophoresis of this material in 1 sodium dodecyl sulfate, which results in disaggregation of the protein and total loss of catalytic activity indicates that at least eight components are present in the active enzyme preparation. Whether these enzymes were present together in the native state as a regulatory unit at the membrane or whether they have associated during the course of purification remains to be determined, particularly since solubilized guinea pig brain Na-K-ATPase is reported to have a molecular weight of 775,000.
Tissues of the rat were removed after decapitation and immediately homogenized in 0.2 M Tris-HC1 (pH 7.4), containing 0.25 M sucrose and 1 mM EDTA-MgCI2 (Levey, 1970). Lubrol-PX was added to the buffer, as will be indicated, to solubilize the enzyme. Protein was determined either by absorbance at 280 nm or by the method of Lowry et a/. (1951). The detergent formed a precipitate during color development in the latter procedure which did not interfere if removed by centrifugation before spectrophotometry. Phospholipid analysis was by the method of Skipski and Barclay (1969). Acrylamide gel electrophoresis was performed by the method of Davis (1964) and electrophoresis in sodium dodecyl sulfate by the technique of Lenard (1970). Lubrol-PX was a gift of Imperial Chemical Industries. The 1T-labeled detergent was generously provided by Dr. Gerald S. Levey. Ficoll and Blue Dextran were obtained from Pharmacia, Bio-Gel A-15m (agarose) from Bio-Rad Labs and DE-52 (diethylaminoethylcellulose) from Whatman. Beef heart cAMP phosphodiesterase, thyroglobulin, and pyruvate kinase were obtained from Sigma. Nucleotides were from P-L Laboratories. All other chemicals were also reagent grade and were obtained from various commercial sources. Results Several tissues of the rat were examined for Lubrol-solubilized adenylate cyclase activity. As seen in Table I, brain yielded the largest amount of solubilized enzyme. The concentration of adenylate cyclase in brain was 13 times that in heart and 27 times that in liver. The enzyme in all of these tissues could be readily solubilized at 0.02, 0.1, and 0.2 M Lubrol. On the basis of the data in Table 11, 0.1 and 0.2 M Lubrol gave a higher yield of solubilized brain enzyme as judged by specific activity of the supernate of the 105,OOOgand 165,OOOg fractions, Thereafter 0.1 M Lubrol was used to solubilize the enzyme. Stirring the enzyme for up to 6 hr in several Lubrol concentrations did not increase the degree of solubilization as judged by activity remaining in the 165,0001: supernatant.
RAT BRAIN ADENYLATE CYCLASE
TABLE I : Distribution
and Solubilization of Adenylate Cyclase in Brain, Liver, and Heart of the Rat.a
Effects of Fluoride and Calcium on Particulate and Soluble Adenylate Cyclase from Rat Brain.
TABLE 111:
Adenylate Cyclase Act. (nmol of cAMP/mg of Protein per 15 min)
Adenylate Cyclase Activity Homogenate Brain Liver Heart
z
105,OOOg Supernatant
Solubilized
6.49 0.27 0.61
58 64 72
11 .26b 0.42 0.85
Tissue of the rat, 1 g/lO ml of buffer, was homogenized in 0.2 M Tris-HC1 (pH 7.4), 1 mM EDTA, 0.25 M sucrose, 0.02 M Lubrol-PX, and 1 mM dithiothreitol, and centrifuged at 10,OOOg for 15 min. The supernatant therefrom was recentrifuged at 105,OOOg for 2 hr and assayed for enzyme activity. All values in nmol of cAMP/mg of protein per 15 min. a
The fate of Lubrol was examined by following the efflux of l4C-labeled Lubrol during dialysis. Half of the labeled Lubrol was removed by overnight dialysis against two changes of 500 volumes each of buffer. The remainder of the detergent was removed by ion-exchange chromatography during subsequent purification of the enzyme. The Lubrol-solubilized brain enzyme was compared to the particulate enzyme for sensitivity to F- and Ca2+. Solubilization does not alter the sensitivity of brain cyclase to F- stimulation (Table 111). Both the particulate enzyme isolated without detergent as a 10,OOOg pellet (McCune et al., 1971) and the solubilized enzyme in the 105,OOOgsupernatant can be stimulated with 2 mM NaF. The qualitative response to Ca*+, however, is modified. Ca2+ stimulated the enzyme in the particulate state but inhibited it after solubilization. To evaluate the degree of solubilization the 105,OOOg supernatant was filtered through 0.22 p Millipore filters. All of the enzyme activity was found in the filtrate, further demonstrating that the adenylate cyclase was removed from its resident site in the membrane. The solubilized enzyme was subjected to gel filtration on Bio-Gel A-15m on a 1.4 X 90 cm column in 0.1 M Tris-HC1
TABLE 11: Effect
of Lubrol-PX Concentration on Brain Adenylate Cyclase Solubilization.a Lubr ol -PX
Homogenate 10,OOOg supernatant l0,OOOg pellet 105, OOOg supernatant 105, OOOg pellet 165, OOOg supernatant 165, OOOg pellet
0.02 M
0.1
M
0.2 M
10.80 10.5 15.2 2.7 26.5 1.8 19.2
8.4 8.6 3.8 10.6 5.0 11.7 9.0
8.6 8.6 10.6 9.4 2.3 10.6 11.0
'Brain was homogenized in the
buffer described in Table I containing, in addition, Lubrol-PX, as indicated ; dithiothreitol was omitted. nmol of cAMP/mg of protein per 15 min.
Membranes' Plus 2 mM NaF Plus 0.025 m~ CaCl,
0.418 i 0.051* 0.610 i 0.035
